PROJECT SUMMARY/ABSTRACT Pancreatic cancer presents a significant public health problem due to its short median survival of six months and projections that deaths from pancreatic cancer will exceed those occurring from breast and colon cancer by 2030. Diagnosing pancreatic cancer at an early stage in patients would enable more efficient and effective intervention, but is hampered by a lack of diagnostic tools. This lack of diagnostic and screening strategies is especially poignant for individuals with two or more first-degree relatives with pancreatic cancer, who have a 9- to 32-fold increased risk. Novel diagnostics would also improve detection of metastases and discrimination between pancreatic cancer and benign pathologies, such as chronic pancreatitis. In addition, improved surveillance methods enable more effective therapeutic intervention upon detection of relapse. Therefore, there remains a pressing need for diagnostic and staging strategies for pancreatic cancer. As such, my ultimate goal is to establish an independent pancreatic cancer research laboratory at a major medical center with an active pancreatic cancer patient program, where I can strategically focus on glycosylation changes as biomarkers of disease and mediators of malignancy. If awarded, the K99/R00 career award will facilitate my transition to research independence, enhance my training in mouse models of disease and mass spectrometric techniques, as well as augment my proficiency at developing clinical-grade diagnostic assays and designing clinical trials under the tutelage of my primary mentor, Dr. David Tuveson. This two-year transition period will also enable extensive training in multiple reaction monitoring methodology for the mass spectrometric-based quantification of biomarker candidates in sera under the guidance of my co-mentor, Dr. Darryl Pappin. The network of great investigators at Cold Spring Harbor Laboratory and established collaborations will enhance my training, accelerate completion of the mentored phase of my proposal, and help me gain the knowledge and experience needed to transition into an independent faculty position. The mentored phase of my research proposal will focus on designing diagnostics for pre-invasive pancreatic cancer and developing better models of pancreatic cancer. During the independent phase, I will identify glycosylation changes during pancreatic cancer progression, and determine their functional relevance to pancreatic cancer. The glycan epitope CA19-9 is the only biomarker for pancreatic cancer. While CA19-9 can be used to follow treatment response, it cannot be used for early detection because it cannot distinguish between inflammatory conditions, such as pancreatitis, and pancreatic cancer. I hypothesize that modifying the manner in which this biomarker is evaluated will enhance its diagnostic utility. Specifically, since CA19-9 is a modification found on many proteins, the test can be altered to detect the presence of CA19-9 on tissue- and disease- specific proteins. Preliminary data using mouse and human pancreatic organoids and patient samples demonstrate that there are CA19-9 carriers specific to malignancy. In Aim 1, I will define CA19-9 carriers that correctly diagnose blinded patient samples (K99 phase). In Aim 2, I will use a mouse model of pancreatic cancer with inducible CA19-9 expression to interrogate the functional role this glycan plays in pancreatic cancer initiation and metastasis, and to use the assays developed in Aim 1 to follow disease progression (K99/R00 phases). In Aim 3, I will investigate the role of glycosyltransferases in pancreatic tumorigenesis (R00 phase). These studies will focus on GCNT1 and GCNT3, which are up-regulated during pancreatic cancer progression. I will use gene editing to ablate these enzymes in organoids followed by the investigation of proliferation and invasion defects in vitro and in vivo, as well as the identification of changes in glycosylation patterns. This research focus will create a multi-modal platform for future research, accelerate progress in this field as it relates to diagnosis and therapeutic targets, and enable my transition to research independence. My research experience and achievements to date are indicative of my potential to succeed in becoming an outstanding independent investigator in pancreatic cancer. This proposal presents a career development plan to transition from highly specialized mentored research focused on developing diagnostic tools to independent research at a major medical center in direct support of pancreatic cancer patients.